INTRODUCTION
Banana is commonly grown in plantations as a monocrop, or in mixed cropping systems, under tropical and subtropical conditions. In mixed and high density cropping systems (Robinson and Nel, Reference Robinson and Nel1988; Rodrigo et al., Reference Rodrigo, Stirling, Teklehaimanot and Nugawela1997; Reference Senevirathna2001), banana may experience mutual shading, and so benefit from changes in microclimate such as small reductions in photosynthetic photon flux density (PPFD), air temperature, turbulence and evaporative demand (Barradas and Fanjul, Reference Barradas and Fanjul1986). One of the most common companion crops grown with rubber in Sri Lanka is banana. Previous studies showed that banana growth improved when intercropped at high densities with rubber, with an increase in light interception and radiation use efficiency, possibly due to the benefits conferred by greater mutual shading in dense canopies (Rodrigo et al., Reference Rodrigo, Stirling, Teklehaimanot and Nugawela1997; Reference Senevirathna2001). Other studies have reported negative impacts of shade on growth and photosynthetic productivity of banana (Israeli et al., Reference Israeli, Schwartz, Plaut and Yakir1996; Robinson and Nel, Reference Robinson and Nel1988).
There is some evidence of acclimation of banana to maximize light capture under low light conditions; for example, banana grown in shade has thinner leaves and increased chlorophyll content (Israeli et al., Reference Israeli, Schwartz, Plaut and Yakir1996). Such acclimation improves the efficiency of light absorption and utilization by leaves (Anderson and Osmond, Reference Anderson, Osmond, Kyle, Osmond and Arntzen1987). However, there are no reports of shade acclimation in terms of dry matter partitioning of banana.
Although numerous studies exist dealing with high temperature effects on banana, little is known of photoinhibition response to high radiation. Plants grown under drought conditions showed a decline in net photosynthesis and F v/F m compared to irrigated plants, indicating a drought-driven photoinhibition in banana leaves (Thomas and Turner, Reference Thomas and Turner1998; Reference Thomas and Turner2001). Since high PPFD has been shown to cause photoinhibition in a range of tropical species (Senevirathna et al., Reference Senevirathna, Stirling and Rodrigo2003; Valladares and Pearcy, Reference Valladares and Pearcy1997), similar effects may be expected for banana, given the potentially high light environments in which it is grown. In such environments, the strong radiation-induced depression of photosynthesis may serve as a protective mechanism to avoid photoinhibitory damage.
All the shade experiments on banana so far reported have involved the use of artificial shades (e.g. Israeli et al., Reference Israeli, Schwartz, Plaut and Yakir1996). Artificially manipulated shade reduces the incident PPFD but has little effect on spectral quality with no changes in the red to far-red ratio, unlike that observed in the understorey of natural vegetation where sunflecks contribute significantly to daily CO2 assimilation (Valladares and Pearcy, Reference Valladares and Pearcy1997). In order to address the limitations of earlier shade studies, we assessed the effects of different shade levels on photosynthetic performance and shade acclimation of banana using natural shade provided by the canopies of rubber plantations.
MATERIALS AND METHODS
Experimental site
The experiment was conducted on the Dartonfield estate of the Rubber Research Institute of Sri Lanka (RRISL), at Agalawatta (6°32′N, 80°09′E, alt. 12 m asl), in the district of Kalutara of the low country wet zone of Sri Lanka. The soil is an Ultisol, with a silty-clay-loam texture and strong brown to yellowish-red in colour (Senevirathna et al., Reference Senevirathna, Stirling and Rodrigo2003; Yogaratnam, Reference Yogaratnam, Peris and Fernando1983).
Experimental design
The experiment comprised four shade treatments, i.e. 0, 33 ± 1.0, 55 ± 0.5 and 77 ± 0.7% reduction in full sunlight and hereinafter referred to as unshaded control, low, medium and high shade treatments, respectively. Natural shade was imposed by selecting existing mature rubber plantations, and the different shade levels were achieved as a result of the age and inter-row spacing of rubber, and by selective pruning of branches of the mature rubber trees (Senevirathna, Reference Senevirathna2001). An open area close to the shaded sites was selected for the unshaded control treatment. Shade levels were monitored by a ceptometer (Delta-T Devices Ltd, Cambridge, UK) and by installing two solarimeters per treatment plot fixed above the canopies of banana plants and connected to the data logger (21 X, Campbell Scientific Ltd, Loughborough, UK) of the weather station (Campbell Scientific Ltd). A nested model was used as the experimental design, and each plant was considered as a single tree plot.
Field establishment and crop husbandry
The most common variety of banana in the wet zone of Sri Lanka, Embul, which belongs to the triploid genome group AAB, was selected for the study. Pre-treated rhizomes with ca. 0.3 m long pseudostems (25 per treatment) were planted in single rows between the rows of mature rubber, keeping an intra-row spacing of 3.0 m. In the unshaded control treatment, banana was planted at an intra- and inter-row spacing of 3.0 m and 4.0 m, respectively. To avoid competition with the roots of the mature rubber trees used to provide natural shade and to minimize variability in rooting conditions, all experimental plants were planted in soil-filled pits lined with a double layer of polythene film of gauge 500 (Musajee Pvt. Ltd., Colombo, Sri Lanka). Soil pits were excavated to a depth of 1 m and to a diameter of 1.8 m. All particles greater than 15 cm3 were removed from the excavated soil, and the pits were refilled according to the original soil profile. Fertilizer was applied according to recommendations, and manual weeding was undertaken when required (Senevirathna, Reference Senevirathna2001).
Leaf gas exchange
Carbon dioxide assimilation rates were measured for a healthy, green, recently expanded third leaf in the canopy in each treatment at about 6 months after planting (MAP) together with the PPFD incident on leaves, using a LI-6200 photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Three replicate plants were selected from each treatment and gas exchange was measured at three intervals in the day, i.e. morning (08:30–10:30 hours), midday (11:00–13:00 hours) and evening (15:00–17:00 hours). Instantaneous measurements of leaf gas exchange were made on bright sunny days (maximum PPFD ca. 2100 μmol m−2 s−1) and on overcast days (maximum PPFD ca. 1200 μmol m−2 s−1) depending on the weather conditions of the experimental site.
Chlorophyll fluorescence
In order to determine the effects of daily incident PPFD levels on photosynthetic capacity of leaves under different light levels, the ratio of variable fluorescence (F v), i.e. the difference between the maximal (F m) and initial (F o) fluorescence (F m − F o), to maximal (F m) fluorescence, F v/F m, was measured at two-hour intervals between 07:00 and 18:00 hours using a Plant Efficiency Analyser (PEA) (Hansatech Instruments Ltd., Kings Lynn, UK). Prior to each set of F v/F m measurements, leaves from five replicate plants in each treatment were dark adapted for 25 min using the leaf clips provided. The saturating light intensity of the PEA (flash used to reduce fully the primary electron acceptor) was adjusted to 50%, as determined for banana leaves during preliminary experiments (Senevirathna, Reference Senevirathna2001). Measurements were taken on clear days (maximum PPFD of ca. 2100 μmol m−2 s−1) and overcast days (maximum PPFD of ca. 1200 μmol m−2 s−1).
Changes in Fv/Fm of artificially shaded and fully exposed leaves
Leaves in the unshaded control were artificially shaded in order to determine the extent to which short-term shading accounted for alleviation of photoinhibition in the unshaded leaves under field conditions. The most recently expanded third leaf from five plants with leaves in the same plane of projection was selected in order to minimize any effects of leaf age and position on treatment response. One part of the middle of the lamina was shaded artificially by placing a neutral white filter paper at a distance of ca. 5 cm above the upper leaf surface throughout the day. Measurements of F v/F m were made from the shaded portion and an adjacent portion of the same half of the lamina, which was exposed to direct insolation. Prior to each set of F v/F m measurements, leaves were dark adapted for a period of 25 min using leaf clips. F v/F m was measured every two hours from 07:00 to 18:00 hours together with measurements of incident PPFD.
Analysis of leaf chlorophyll
Chlorophyll a/b ratio and total chlorophyll analyses were carried out for plants grown in the four shade treatments in order to determine the effect of shade on leaf chlorophyll. Lamina pieces of the most recently expanded third leaf from five plants in each treatment were harvested early in the morning at ca. 12 MAP. In the laboratory, 5 g of fresh leaf tissue was taken and cut into small pieces, ground with 80% aqueous acetone and the pigments were extracted at room temperature (27 °C) for about two hours. The extracts were then centrifuged at 500 g for five min, and the resultant supernatant separated from the precipitate by filtering through a Whatman No. 1 filter paper. The absorbance of the supernatant was measured using a spectrophotometer (M 330, Camspec, Cambridge, UK) at wavelengths 645 and 663 nm, using 80% acetone as the blank, and total chlorophyll and chlorophyll a/b were calculated (Hipkins and Baker, Reference Hipkins and Baker1986).
Growth analysis
Using destructive sampling, both above- and below-ground dry matter was measured at seven MAP using mother plants and again at 17 MAP using the first ratoon crop, which is produced by the rhizome of mother plants. Five plants were randomly selected from each treatment for the growth analysis. Selected plants were completely uprooted and the total fresh weights of the components (leaves, pseudostem, rhizome and roots) were measured separately immediately after harvest. A sub-sample of 40% of the total fresh weight from each component was used for the dry matter determination (Rodrigo et al., Reference Rodrigo, Stirling, Teklehaimanot and Nugawela1997). Samples were dried at 80 °C to a constant weight and total dry weights recorded. The area of each leaf was estimated according to the equation, leaf area = maximum length × maximum width × 0.755, after Rodrigo et al. (Reference Rodrigo, Stirling, Teklehaimanot and Nugawela1997). Leaf area ratio (LAR, m2 kg−1), specific leaf area (SLA, m2 kg−1) and leaf weight ratio (LWR, kg kg−1) were calculated as described by Hunt (Reference Hunt1978).
Data analysis
Plant dry weights, LAR, SLA, LWR, diurnal changes in CO2 assimilation and F v/F m were analysed with the nested procedure of SAS software (SAS Institute Inc., Cary, NC, USA). The procedure analysis of variance (ANOVA) was used for data analysis. The mean separation of treatments was performed with Duncan's multiple range test for the comparison of treatment effects.
RESULTS
CO2 assimilation
PPFD incident on shaded plants was low throughout the day compared to the unshaded control, both on sunny and overcast days (Figure 1a,b). PPFD reached a mean maximum of 1801 (± 171) and 810 (± 252) μmol m−2 s−1 under clear sky and overcast conditions, respectively. Corresponding with the diurnal variation in PPFD, CO2 assimilation rates were generally lower in the shade treatments than in the control (Figure 1c,d).
Diurnal changes in photosynthetic photon flux density (PPFD) on (a) sunny and (b) overcast days, and corresponding CO2 assimilation rates on the same (c) sunny and (d) overcast days for banana in the unshaded control, low medium and high shade treatments where the reduction in incoming radiation was 0, 33, 55 and 77%, respectively. Each value is the mean of six measurements ± s.e. Morning, midday and evening measurements were taken between 08:30 and 10:30 hours, 11:00 and 13:00 hours, and 15:00 and 17:00 hours, respectively.
The highest CO2 assimilation rate (21.5 μmol m−2 s−1) under clear sky conditions was recorded in the unshaded control in the morning with assimilation rates significantly higher (p < 0.001) than in shade treatments. A similar pattern of CO2 assimilation was observed in all the shade treatments, even under overcast conditions (Figure 1d). Furthermore, the highest shade treatment showed the lowest CO2 assimilation rates throughout the day, both under sunny and overcast conditions (Figure 1c,d).
Chlorophyll fluorescence
The ratio of F v/F m showed a clear midday depression and recovery towards evening on both sunny and overcast days, with the midday depression greatest under clear sky conditions coinciding with the diurnal peak in PPFD (Figure 2). The highest and lowest values of F v/F m were recorded in the high shade and unshaded control, respectively, throughout the day and under both sunny and overcast conditions (p < 0.001). Under sunny conditions, F v/F m of the control declined from 0.818 (± 0.003) in the early morning to 0.757 (± 0.006) in the afternoon (13:00–14:00 hours). Values of F v/F m varied little during the day in the highest shade treatment with a range of between 0.84 and 0.82 both under clear sky and overcast conditions (Figure 2c,d). Low and medium shade produced more or less similar results, under both sunny and overcast conditions, except between 13:00 and 16:00 hours on the sunny day when the low shade treatment showed a greater depression in F v/F m (p < 0.01) than the medium shade plants (Figure 2c,d).
Diurnal changes in photosynthetic photon flux density (PPFD) on (a) sunny and (b) overcast days, and corresponding F v/F m ratios on the same (c) sunny and (d) overcast days for banana in the unshaded control, low medium and high shade treatments where the % reduction in incoming radiation was 0, 33, 55 and 77%, respectively. Each value is the mean of five measurements ± s.e.
The midday decline in F v/F m was significantly less (p < 0.001) when leaves in the control were artificially shaded and F v/F m recovered rapidly in these leaves at the end of the day. Artificial shading reduced incoming radiation by 75–80% and increased F v/F m maximally by 16% at 13:00–14:00 hours (Figure 3a,b). Artificial shading lowered leaf temperature too, by a maximum of 2 °C (Figure 3c).
Diurnal changes in (a) photosynthetic photon flux density (PPFD), and corresponding (b) F v/F m ratios and (c) leaf temperature of banana leaves of unshaded and artificially shaded parts of leaves in the unshaded control treatment. Each value is the mean of five measurements ± s.e. Shades were provided by filter paper positioned 5 cm above the upper surface of leaves.
Partitioning of dry matter
Total dry matter (TDM) production at seven MAP decreased with increasing shade (Figure 4a). However the first ratoon crop (17 MAP) showed a similar TDM production in the control and medium shade treatments, which were both significantly higher (p < 0.01) than the TDM of the low and high shades (Figure 4a). At seven MAP, the unshaded control produced 3.58 (± 0.72) kg dry matter, which was on average three-times greater than in shade treatments (Figure 4a). Furthermore, a linear relationship between cumulative incident radiation and cumulative dry matter production was observed for the 17-month period (Figure 4b, r 2 = 0.848). SLA increased (p < 0.01) with increasing shade at seven MAP; at 17 MAP, the high shade treatment showed apparently a higher (p < 0.01) SLA over the other shade treatments (Table 1). Similar to SLA, treatments effects on LAR were most prominent at seven MAP when LAR increased significantly with shade (p < 0.001). In the ratoon crop, no consistent difference between treatments was observed with the exception of the high shade treatment where LAR was always greater (p < 0.01). LWR of banana decreased from seven to 17 MAP, but no significant treatment effect was observed (Table 1).
Summary of the dry matter production of banana where (a) is the total dry matter per plant at 7 (mother crop) and 17 (first ratoon crop) months after planting and (b) is the relationship between cumulative dry matter production and cumulative incident radiation for the period in the unshaded control, low medium and high shade treatments where the % reduction in incoming radiation was 0, 33, 55 and 77% respectively. Data represent the mean of five replicate plants ± s.e.
Summary of specific leaf area (SLA), leaf area ratio (LAR) and leaf weight ratio (LWR) of banana at seven (mother crop) and 17 (first ratoon crop) months after planting (MAP) in the unshaded control, low medium and high shade treatments where the % reduction in incoming radiation was 0, 33, 55 and 77%, respectively. Data represent the means of five replicate plants ± s.e.
Chlorophyll content
Total chlorophyll concentrations tended to be higher in the shaded treatments than in the unshaded control but, unexpectedly, chlorophyll concentration was low in the highest shade treatment compared to the low and medium shade treatments (Figure 5a). However, the chlorophyll a/b ratio declined with increasing shade from a maximum of 2.30 (± 0.19) in the control to 1.94 (± 0.02) in the high shade treatment (Figure 5b).
Summary of the chlorophyll analysis of banana leaves at 12 months after planting where (a) is the total and (b) is the a/b ratio of chlorophyll in the unshaded control, low medium and high shade treatments where the % reduction in incoming radiation was 0, 33, 55 and 77%, respectively. Data represent the mean of five replicates ± s.e.
DISCUSSION
Growth and physiological performance of banana grown under shade differed from that of plants grown under full sunlight. Rates of photosynthesis revealed that unshaded plants had the highest assimilation capacity of all groups of plants. Assimilation of CO2 measured under sunny and overcast conditions (Figure 1) indicated that PPFD incident on control plants on bright sunny days was far above the saturation levels; hence, there was little variation in CO2 assimilation between sunny and overcast conditions. Photosynthetic light saturation of banana grown under full sunlight in this research was ca. 1000 μmol m−2 s−1, similar to that observed by Rodrigo et al. (Reference Rodrigo, Stirling, Teklehaimanot and Nugawela2001) under similar tropical conditions. As indicated by the lower F v/F m ratios, excessive PPFD incident on banana leaves, especially during the central hours of the day, resulted in greater dynamic photoinhibition in unshaded plants (Figure 2). Moreover, artificial shading of leaves in the unshaded control treatment showed that even short-term shading helps to alleviate high radiation stress in banana (Figure 3). The rapid recovery in F v/F m towards the end of the day in the unshaded control (Figure 2, 3), suggested that the midday depression in F v/F m was most likely due to dynamic photoinhibition rather than photoinhibitory damage to photosystem II, as observed for other tropical crops (Senevirathna et al., Reference Senevirathna, Stirling and Rodrigo2003). This phenomenon is a photoprotective mechanism rather than photodamage to the photosystem.
Although depression in F v/F m has been recorded in relation to low temperature (Damasco et al. Reference Damasco, Smith, Godwin, Adkins, Smillie and Hetherington1997), reports on high-light-induced photoinhibition of banana are limited compared to other tropical plants (e.g. Ishida et al., Reference Ishida, Nakano, Matsumoto, Sakoda and Ang1999; Senevirathna et al., Reference Senevirathna, Stirling and Rodrigo2003). It was clear that the extent of high-light-induced photoinhibition was limited in plants grown under heavy shade. The reduction in incident PPFD in shaded treatments resulted in a corresponding reduction in TDM (Figure 4a), and this was supported by the linear correlation between cumulative incident radiation and cumulative dry matter production (Figure 4b). The apparent anomaly in TDM of shaded treatments where values were higher in the medium than the low and high shade treatments during the ratoon crop period, i.e. at 17 MAP (Figure 4a) may have been due to the differences in times of initiation of the first ratoon crop under different shade levels. However, cumulative dry matter production at seven and 17 MAP showed similar values in low and medium shade treatments.
Although TDM was reduced under shade conditions, banana showed an ability to acclimate to shade with an increase in SLA with increasing shade (Table 1). The production of thinner leaves under shade is an acclimation to enhance the efficiency of light utilization in shaded environments (Evans and Poorter, Reference Evans and Poorter2001). Increased LAR with increasing shade resulted from an increased ratio of dry matter partitioned to leaves under shade. LWR did not vary much across shade treatments, as both SLA and LAR changed in direct proportion to the density of shade. In addition to the shade acclimations in terms of increased SLA and LAR, the chlorophyll a/b ratio of leaves decreased under shade (Figure 5). This reflected the increased association of chlorophyll with the light harvesting complexes, which are rich in chlorophyll b (Evans and Poorter, Reference Evans and Poorter2001), in order to increase the efficiency of light harvesting in shade environments.
Since incident PPFD on unshaded plants was far above the saturation levels for photosynthesis, dynamic photoinhibition was greatest in plants that received full sunlight on bright sunny days revealing that photoprotection occurs in banana without damage to the photosystems. Although this study demonstrated that banana has the ability to acclimate to shade and to reduce dynamic photoinhibition under shade, the PPFD incident on the plants even in the lowest shade was not high enough to reflect such benefits in terms of dry matter accumulation. Therefore, mild shade in which the level of PPFD is high enough to saturate CO2 assimilation and low enough to induce shade acclimation will help to optimize the photosynthetic productivity of banana.
Acknowledgements
We wish to thank the Rubber Research Institute of Sri Lanka for providing land for the experiment and staff at the institute for valuable assistance in the field. Our thanks are extended to Dr (Mrs) Wasana Wijesuriya, biometrician at the Rubber Research Institute of Sri Lanka for her great assistance in analysing data. This publication is an output from the project, R7212 of Plant Science Research Programme funded by the UK Department for International Development (DFID) for the benefit of developing countries. The views expressed are not necessarily those of DFID.
